Strains of [PSI(+)] are distinguished by their efficiencies of prion-mediated conformational conversion.

Abstract

Yeast prions are protein-based genetic elements that produce phenotypes through self-perpetuating changes in protein conformation. For the prion [PSI(+)] this protein is Sup35, which is comprised of a prion-determining region (NM) fused to a translational termination region. [PSI(+)] strains (variants) with different heritable translational termination defects (weak or strong) can exist in the same genetic background. [PSI(+)] variants are reminiscent of mammalian prion strains, which can be passaged in the same mouse strain yet have different disease latencies and brain pathologies. We found that [PSI(+)] variants contain different ratios of Sup35 in the prion and non-prion state that correlate with different translation termination efficiencies. Indeed, the partially purified prion form of Sup35 from a strong [PSI(+)] variant converted purified NM much more efficiently than that of several weak variants. However, this difference was lost in a second round of conversion in vitro. Thus, [PSI(+)] variants result from differences in the efficiency of prion-mediated conversion, and the maintenance of [PSI(+)] variants involves more than nucleated conformational conversion (templating) to NM alone.

Fig. 1. Analysis of the amount of nonsense suppression and soluble Sup35 in [PSI+] variants. (A) Five-fold serial dilutions and growth of [PSI+] variants on rich media (YPD, 30°C, 4 days) and media lacking adenine (SC–Ade, 30°C, 14 days). (B) Quantitation of translational read through of [PSI+] variants. (C) Dot-blot analysis of Sup35 solubility in [PSI+] variants. Proteins from the top fraction of each sucrose cushion were normalized, serially diluted in 2-fold increments and applied to a PVDF membrane. Top, reactivity with a Sup35-specific antibody (). Bottom, membrane stained with a non-specific protein stain, Ponceau-S. The amount of total protein per column is indicated at the top.

Fig. 3. Sup35PSI+ from different [PSI+] variants differ in NM conversion efficiency. (A) Effect of sucrose cushion P3 proteins isolated from several [PSI+] variants on NM conversion. Sucrose pellet P3 was used at a 100-fold dilution. Curves are drawn to indicate the likely data trends and are not correlated sigmoidal fits. (B) Extent of NM degradation during the conversion in the presence of different P3 pellets as shown in (A). Coomassie Blue R-250 staining of proteins separated by SDS–PAGE. The arrow shows migration of full-length NM. The positions and molecular weights of pre-stained marker proteins are shown on the right. Minor faster migrating bands present in all lanes are NM degradation products that co-purified from E.coli (data not shown). (C) Comparison of the amount of Sup35 in each P3 preparation. Pellet fractions were serially diluted in 2-fold increments as indicated and separated by PAGE. Sup35 was detected with NM-specific antiserum (). (D) Effect of different dilutions of P3 preparations on the conversion efficiency of Sup35PSI+ from a strong and weak [PSI+] variant. P3 from strong [PSI+] was diluted either 100- or 200-fold and P3 from weak variant 13 was diluted 10- or 100-fold as indicated. (E) Estimation of the amount of Sup35 present in each P3 pellet fraction. Purified NM, which was serially diluted as indicated and subjected to PAGE, served as a protein standard. NM and Sup35 were detected by immunoblotting with NM-specific antiserum (). Since P3 preparations from all [PSI+] variants have similar amounts of Sup35 (C), only the strong [PSI+] P3 pellet fraction is shown (upper blot). A darker exposure of the strong [PSI+] samples of (C) is shown here, because the signals were not visible without a longer exposure. (F) Comparison of the conversion efficiency of Sup35PSI+ from a strong [PSI+] variant with that of spontaneously formed NM fibers. P3 from strong [PSI+] was diluted 25-fold and spontaneously formed NM fibers were diluted 1000-fold; thus, the ratio of Sup35PSI+ or NM in fibers to soluble NM was ∼1:1000.

Fig. 4. Structural and biochemical characterization of fibers converted in the presence of Sup35PSI+ from different variants. (A) AFM images taken in tapping mode of fibers formed in the presence of Sup35PSI+ from strong or weak variants or with an identically prepared fraction from a [psi–] strain. The white bar represents 100 µm. (B) Second derivative attenuated total reflectance infrared spectra of NM fibers converted with strong [PSI+], weak [PSI+] (13 and 21) and [psi–] lysates. Second derivative spectra show negative deflections that correspond to positive absorbance maxima in primary spectra and provide a means of better resolving small differences between infrared spectra. For comparison, the spectra of hemoglobin (predominantlyα-helix) and PrPSc (mixed α-helix and β-sheet) are included. The amide I region is shown, which is sensitive primarily to the secondary structure of the polypeptide backbone. No consistent differences were observed between the four types of NM fiber in either the primary (not shown) or second derivative spectra. All of the NM fiber spectra showed predominant negative bands in the region dominated by β-sheet absorbances (∼1616–1640 cm–1); however, the pattern of these probable β-sheet bands was distinct from that of PrPSc. The other major NM fiber band was centered at ∼1660 cm–1, a wavenumber that is inconsistent with either β-sheet or α-helix but consistent with the presence of turn structure(s). (C) Determining whether the characteristic conversion efficiencies of Sup35PSI+of variants can be serially passaged to a second NM preparation. Sucrose P2 pellets from [psi–] and two [PSI+] variants (strong or weak 21) were used to prepare NM fibers that were subsequently diluted 1000-fold into another preparation of 10 µM soluble NM.